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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 鄭石通(Shih-Tong Jeng) | |
| dc.contributor.author | Zong-Yan Liu | en |
| dc.contributor.author | 劉宗晏 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:42:29Z | - |
| dc.date.available | 2025-08-14 | |
| dc.date.copyright | 2020-08-28 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78108 | - |
| dc.description.abstract | 植物在遭受到傷害訊號時，會產生胜肽激素以參與植物防禦機制並觸發相關基因的表達，從而保護植物免於受到病原體的入侵等相關逆境。MicroRNA (miRNA) 是由20-24個核苷酸所組成之非編碼RNA，其藉由與互補序列之鹼基形成配對來降低其目標基因的表現。但是，植物胜肽激素和miRNA之間的關係鮮少被研究。本篇研究中，著重於分析甘藷栽培種臺農57號 (Ipomoea batatas cv. Tainung 57) 中，受到甘藷胜肽激素hydroxyproline-rich glycopeptides (HypSys) 所調控的調控miRNA。因甘藷葉片在機械性損傷後會由IbpreproHypSys剪切而成具有功能之IbHypSys。因此，本實驗以過量表達IbpreproHypSys的轉殖株 (OE) 及野生型 (WT) 甘藷，進行small RNA 次世代定序和轉錄組 (transcriptome) 分析。程式預測的方法則是分析系統葉片中OE在受傷2小時前後的miRNA表現量。分析WT與OE之結果顯示出，共有13種miRNA包含miR166、miR398、miR399、miR403和一些新穎miRNA會因受到傷害而下調表現量。綜合比較transcriptome與miRNAs得知，這些miRNAs是會由IbHypSys胜肽進行調控。此外，miRNA的目標基因則是透過psRNAtarget和CleaveLand4兩個不同的分析方式進行預測。因此找出miR398及其目標基因IbSAD2作為後續研究項目。藉由即時定量PCR的結果顯示，在OE的甘藷之中，IbSAD2的表達明顯高於野生型，表示出IbSAD2是受到IbHypSys所誘導而成。並且在菸草中進行agroinfiltration 確認IbSAD2是會受到miR398辨識並切割。綜上所述，甘藷受到傷害逆境後，會促進IbHypSys產生， IbHypSys則會降低miR398的表現，使IbSAD2的表現增加，以進行傷害防禦反應。 | zh_TW |
| dc.description.abstract | Upon wounding, plants produce peptide hormones to engage in plant defense and trigger the expression of defense genes to protect plants from pathogens. MicroRNAs (miRNAs) are non-coding RNA with 20-24 nucleotides and cleave their targets by base pairing with complementary sequences. However, the relationship between plant peptide hormones and miRNAs is seldom investigated. In this study, the regulation of hydroxyproline-rich glycopeptides (HypSys), a plant peptide hormone, on microRNAs were analyzed in sweet potato (Ipomoea batatas cv. Tainung 57). IbHypSys is cleaved from IbpreproHypSys after wounding. The small RNA deep sequencing and transcriptome data from transgenic sweet potato overexpressing IbpreproHypSys (OE) under wounding for 2 hours were analyzed. After these sequencing data from the systemic leaves of wildtype (WT) and OE were compared, 13 miRNAs, comprising of miR166, miR398, miR399, miR403, and some novel miRNAs, were obtained to be down-regulated by wounding. Comprehensive analysis of the transcriptome data and miRNAs suggested that the expression of miRNAs was affected by IbHypSys peptide. Target genes of miRNAs were also predicted by psRNAtarget and CleaveLand4. The miR398 and its target IbSAD2 was further studied. Results of RT-qPCR indicated that the expression of IbSAD2 was significantly increased in OE than in WT, suggesting the expression of IbSAD2 may be induced by IbHypSys. The recognition of miR398 on its binding site on IbSAD2 was further identified by agroinfiltration, indicating IbSAD2 was a target of miR398. The expression of target gene IbSAD2 might enhance the response of plants to wounding. In conclusion, IbHypSys-induced miRNAs may regulate wounding response, and this research may illuminate wounding defense in sweet potato. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:42:29Z (GMT). No. of bitstreams: 1 U0001-1408202015065100.pdf: 3148664 bytes, checksum: ac961bb5b97a976dab761fdf8e41a154 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員會審定書 i 誌謝 ii 摘要 iii Abstract iv 1 Introduction 1 1.1 Plant Wounding Stresses and Wounding Responses 1 1.2 Plant Peptide Hormones 2 1.3 Hydroxyproline-rich systemins (HypSys) 3 1.4 Biosynthesis pathway of miRNAs in plants 4 1.5 Stress-mediated miRNAs in plants 6 1.6 Prediction of miRNA target genes 6 1.7 Research motivations and objectives 7 2 Materials and methods 9 2.1 Plant materials growth conditions and treatment 9 2.1.1 Sweet potato 9 2.1.2 Tobacco 10 2.2 Process of RNA reverse transcription to cDNA 10 2.2.1 Total RNA extraction 10 2.2.2 RNA Electrophoresis 11 2.2.3 DNase treatment 11 2.2.4 Reverse transcription (RT) 12 2.3 Assay of the gene expression 13 2.3.1 Polymerase Chain Reaction (PCR) 13 2.3.2 Quantitative real time polymerase chain reaction (qRT-PCR) 14 2.3.3 DNA gel electrophoresis 15 2.4 Plasmid construction 15 2.4.1 Gel elution 15 2.4.2 DNA ligation 16 2.4.3 DH5α (E. coli) transformation 16 2.4.4 Plasmid DNA mini extraction 17 2.4.5 DNA sequencing 18 2.4.6 Construction of the vector by restriction enzyme 18 2.5 Agrobacterium-mediated tobacco transformation 18 2.5.1 Preparation of Agrobacterium LBA4404 18 2.5.2 Transformation of Agrobacterium strain LBA4404 19 2.5.3 Agrobacterium-mediated transient expression in tobacco 20 2.6 Next generation sequencing data analysis 21 2.6.1 Small RNA sequencing 21 2.6.2 Raw data cleaning 21 2.6.3 Comparison of the putative primary miRNAs 22 2.6.4 Distinguish the IbHypSys-repressed miRNAs between conserved and novel 24 2.6.5 Prediction of miRNAs target genes 25 3 Results 27 3.1 Construction library of high-throughput small RNA sequencing data 27 3.2 Identification of the conserved and novel IbHypSys-regulated miRNAs 28 3.2.1 Debrief from the small RNA sequencing library 29 3.2.2 Sequence comparison of miRNA candidates and de novo assembly transcriptome 30 3.2.3 Verification of the stem-loop structure in the putative pre-miRNAs 30 3.2.4 miRNAs star strand in the putative pre-miRNAs 31 3.2.5 IbHypSys-repressed putative miRNAs in sweet potato 32 3.3 IbHypSys-repressed miRNAs in sweet potato 33 3.4 Participation of IbHypSys in miR398 regulation 34 3.5 The primary form of sweet potato miR398 35 3.6 The prediction of miR398 target genes 35 3.7 The expression of importin beta-like SAD2 under wounding 37 3.8 Attestation of the relationship between miR398 and importin beta-like SAD2 38 3.9 Importin beta-like SAD2 might contribute to plant defense 39 4 Discussion 41 4.1 The IbHypSys-mediated miRNA candidates of the systemic leaves in sweet potato 41 4.2 Assemble of sweet potato transcriptome sequencing data for miRNAs prediction 42 4.3 Expression level of miR398 in sweet potato 44 4.4 Software for predicting miR398 target gene 45 4.5 Function of miR398 target gene importin beta-like SAD2 47 5 Conclusion 50 6 Figures and figure legends 51 Table 1. Results of small RNA deep sequencings from the systemic leaves of sweet potato 51 Table 2. The unique miRNAs repressed in the systemic leaves of transgenic plant overexpressing IbpreproHypSys 52 Table 3. Prediction of miR398’s target genes 54 Table 4. Primers used in this study 55 Description 55 Reverse Transcript 55 Normal cDNA reverse transcript 55 Small RNA reverse transcript 55 Figure 1. The length distribution counts of all small RNA sequencing data 57 Figure 2. Workflow for the prediction of miRNAs and their targets 58 Figure 3. Pre-miR398, pre-miR399, and pre-miR403 secondary structures predicted by mfold. 59 Figure 4. The expression levels of miR398, miR399, and miR403 in the systemic leaves of transgenic plant overexpressing IbpreproHypSys 60 Figure 5. The expression levels of miR398, miR399, and miR403 in the systemic leaves of wildtype sweet potato 61 Figure 6. The expression levels of miR398 in the local and systemic leaves of wildtype and transgenic sweet potatoes 62 Figure 7. Primary form of miR398 in pCAMBIA-2300 63 Figure 8. Agro-infiltration of primary form of miR398 to tobacco (Nicotiana benthamiana) for examining mature miR398 64 Figure 9. Venn diagram of the analysis results from sweet potato degradome by GSTAr and transcriptome data by psRNAtarget 65 Figure 10. The binding site of miR398 in its predicted target gene importin beta-like SAD2 66 Figure 11. The expression of miR398 target gene importin beta-like SAD2 67 Figure 12. Importin beta-like SAD2 binding site in pCAMBIA-1301 69 Figure 13. Agro-infiltration of primary form of miR398 to tobacco (Nicotiana benthamiana) for examining primary miR398 70 Figure 14. The recognition of SAD2 binding site by mature miR398 was confirmed by agroinfiltration in tobacco 71 Figure 15. Comparison of SAD2 amino acid sequences among Ipomoea batatas, Solanum lycopersicum, and Arabidopsis lyrate 72 Supplemental Table 1. The thermodynamic details of miR398, miR399, and miR403 precursor forms calculated by mfold. 73 Supplemental Table 2. miR398 targets predicted by psRNATarget and GSTAr 76 Supplemental Table 3. The primary SAD2 sequence from sweet potato 78 Supplemental Table 4. BLASTP of importin beta-like SAD2 among plants 80 Supplemental Figure 1. The quality scores given by FastQC 81 Supplemental Figure 2. The results of miR398 target genes predicted by CleaveLand4. 83 7 References 84 | |
| dc.language.iso | en | |
| dc.subject | 甘藷 | zh_TW |
| dc.subject | SAD2 | zh_TW |
| dc.subject | miR398 | zh_TW |
| dc.subject | IbHypSys | zh_TW |
| dc.subject | 系統性傷害 | zh_TW |
| dc.subject | SAD2 | en |
| dc.subject | miR398 | en |
| dc.subject | sweet potato | en |
| dc.subject | systemic wounding | en |
| dc.subject | IbHypSys | en |
| dc.title | 傷害逆境下受IbHypSys調控表達的甘藷microRNAs之研究 | zh_TW |
| dc.title | Analysis of IbHypSys-mediated MicroRNAs upon Wounding in Sweet Potato (Ipomoea batatas cv. Tainung 57) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃浩仁(Hao-Jen Huang),洪傳揚(Chwan-Yang Hong),林振祥(Jeng-Shane Lin) | |
| dc.subject.keyword | 甘藷,系統性傷害,IbHypSys,miR398,SAD2, | zh_TW |
| dc.subject.keyword | sweet potato,systemic wounding,IbHypSys,miR398,SAD2, | en |
| dc.relation.page | 95 | |
| dc.identifier.doi | 10.6342/NTU202003423 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-18 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| dc.date.embargo-lift | 2025-08-14 | - |
| Appears in Collections: | 植物科學研究所 | |
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